Printing of fsl constructs

ABSTRACT

Methods of manufacturing cards, strips and multiwell plates for use in diagnostic assays are described. The methods employ the use of synthetic constructs of the generic structure F-S-L (where F is an antigen for a reactive antibody, S is a spacer covalently linking F to L, and L is a lipid) as “ink” in inkjet printers. The cards, strips and multiwell plates provide a number of advantages including efficiency of manufacture and improved accuracy in determining and recording assay results.

FIELD OF INVENTION

The invention relates to a method of printing constructs of the genericstructure F-S-L (where F is a functional moiety, S is a spacercovalently linking F to L, and L is a lipid).

In particular, the invention relates to the use of the method in thefabrication of diagnostic test cards and sticks, microarrays andmultiwell plates.

BACKGROUND ART

Glycomics has emerged with proteomics as an area for development andexploration in the postgenomics era (Blixt et al (2004)). Despite theincreasing awareness of the biological significance of carbohydrates,the study of carbohydrate-protein interactions still encounters muchdifficulty. There is a need for the development of highly sensitive andhigh-throughput methods for identification and binding study ofcarbohydrates recognized by various receptors (Chung-Yi et al (2009)).

The immobilization of glycans on the derivatised surface of substratesis a commonly employed method of fabricating glycan microarrays. Blixtet al (2004) discloses immobilisation of amine functionalised syntheticglycan ligands on N-hydroxysuccinimide (NHS) activated glass slidesusing a custom made robotic printing arrayer. Bovin and Huflejt (2008)have reviewed the use of binding chemistries exploiting amide bondformation. Short spacers are used to reduce non-specific contacts to aminimum. Attachment to a flexible layer of polyethylene glycol on aglass surface is presented as assuring availability of glycan moietiesfor interaction with binding molecules.

The localization of glycans to the surface of substrates in the form ofneoglycolipids has also been employed as a method of fabricating glycanmicroarrays. Chai et al (2003) describe a multiwell-binding assay inwhich neoglycolipids are diluted either in methanol, or in methanolcontaining the carrier lipids egg lecithin and cholesterol. Thedispersions of neoglycolipids are then used to coat the wells of themultiwell plates. Chai et al (2004) describe the bandwise application ofthe dispersions of neoglycolipids by a spray-on technique employing asample applicator comprising a single syringe as applicator (LINOMAT IV,Camag, Switzerland)

Fukui et al (2005) and Huang et al (2006a, 2006b) have each described anon-covalent glycoarray assembly method utilising lipid-linkedsaccharides and oligosaccharides. Both methods employ reductiveamination to produce a lipid-linked saccharide of oligosaccharide(neoglycolipid). In the method of Fukui et al (2005) oligosaccharideswere conjugated to 1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine(DHPE) directly or after mild periodate oxidation. The neoglycolipidswere then applied by jet spray as bands or spots onto nitrocellulosemembranes. In the method of Huang et al (2006a, 2006b) the reaction toproduce lipid-linked saccharides uses an excess of saccharides in orderto exhaust the tetradecylamine employed in the reaction. Thelipid-linked saccharides were then applied to multi-well high bindingpolystyrene plates.

Liu et al (2006) describe the preparation of neoglycolipids fromN-aminooxyacetal DHPE (AOPE) by a chemoselective oxime-ligation reactionwith reducing sugars. The binding of the neoglycolipids by antibodiesand lectins was assayed by an enzyme-linked immunosorbent assay (ELISA)in plastic microwells as described by Chai et al (2003). In thesestudies the neoglycolipids were incorporated into liposomes for arrayingand spotted onto nitrocellulose membranes or robotically arrayed ontonitrocellulose-coated glass slides.

Palma et al (2006) and Campanero-Rhodes et al (2007) describe thepreparation of arrays of natural and synthetic glycolipids andneoglycoplipids by printing on nitrocellulose-coated glass slides usinga non-contact piezoelectric arrayer (PIEZORRAY, Perkin-Elmer, UnitedKingdom). Liu et al (2007) also describes the use of this non-contactpiezoelectric arrayer. The arrayer employs an assembly containing fourPIEZOTIP™ dispensers to dispense sub-nanolitre to nanolitre volumes with20 to 25 μm accuracy and precision.

It is an object of the invention to provide an improved method for thelocalisation of functional moieties, including glycans, to the surfaceof substrates.

It is an object of the invention to provide a method of fabricatingdiagnostic test cards and sticks, microarrays and multiwell plates.

It is an object of the invention to provide templates for use in thefabrication of diagnostic test cards and sticks, microarrays andmultiwell plates microarray formats by the method that improve accuracyand reliability of assay results.

These objects are each to be read disjunctively with the object to atleast provide the public with a useful choice.

STATEMENT OF INVENTION

In a first aspect the invention provides a method of localising afunctional moiety (F) to at least one discrete area on a surface of asubstrate including the step of:

-   -   Propelling droplets of a dispersion of a synthetic construct of        the structure F-S-L from a plurality of orifices onto the        surface of the substrate;        where:    -   S is a spacer; and    -   L is a lipid.

Preferably, F is biotin, a glycan or a peptide.

In a first preferment of the first aspect of the invention, the at leastone discrete area is in the shape of a symbol. More preferably, the atleast one discrete area is in the shape of a symbol readable by opticalcharacter recognition (OCR) apparatus. Most preferably, the at least onediscrete area is in the shape of a symbol comprising one or morealphanumeric characters. In a second preferment of the first aspect ofthe invention, the at least one discrete area is a pattern comprising acombination of indicia in which the dispersion of a synthetic constructis present at different densities (amount per unit area). The first andsecond preferments of this aspect of the invention are not mutuallyexclusive.

Preferably, the substrate is selected from the group consisting of:derivatised silica gel (e.g. C₈ or C₁₈), nitrocellulose, coated paper,silica gel or uncoated paper. More preferably, the substrate is selectedfrom the group consisting of: coated paper or uncoated paper.

Preferably, the method is a non-impact method of printing. Morepreferably, the propelling droplets from a plurality of orifices is froma plurality of orifices located in a monolithic print head. Yet morepreferably, the propelling droplets from a plurality of orifices is froma plurality of orifices located in a monolithic print head of an inkjetprinter. Most preferably, the propelling droplets from a plurality oforifices is from a plurality of orifices located in a monolithic printhead of a piezoelectric inkjet printer.

Preferably, the volume of each of the droplets is 1 to 100 picolitres(pL). More preferably, the volume of each of the droplets is 1 to 50 pL.Most preferably, the volume of each of the droplets is 1 to 5 pL.

Preferably, the concentration of the synthetic construct in thedispersion is 1 μmolar (μM) to 10 mmolar (mM). More preferably, theconcentration of the synthetic construct in the dispersion is 10 μM to10 mM. Most preferably, the concentration of the synthetic construct inthe dispersion is 0.1 to 10 mM.

Preferably, the spacer (S) is selected to provide a construct that isdispersible in water in the absence of organic solvents or detergents ata temperature of 25° C. More, preferably, the synthetic construct of thestructure F-S-L is dispersible in water in the absence of organicsolvents or detergents at a temperature of 25° C. at a concentration ofat least 6 millimolar (mM). More preferably, the synthetic construct ofthe structure F-S-L is dispersible in water in the absence of organicsolvents or detergents at a temperature of 25° C. at a concentration ofat least 12 millimolar (mM).

Preferably, L is a diacyl- or dialkyl lipid. More preferably, L is aglycerophospholipid. Yet more preferably, L is aphosphatidylethanolamine. Most preferably, L is selected from the groupconsisting of: 1,2-O-dioleoyl-sn-glycero-3-phosphatidylethanolamine(DOPE) and 1,2-O-distearyl-sn-glycero-3-phosphatidylethanolamine (DSPE).

In a first preferment of the first aspect of the invention F is aglycan. Preferably, F is a glycan that is an oligosaccharide. Morepreferably, F is a glycan that is an oligosaccharide selected from thegroup consisting of: GalNAcα3(Fucα2)Galβ-; Galα3(Fucα2)Galβ-;GalNα3(Fucα2)Galβ-; Fucα2Galβ-; Galβ4GlcNAβ3(Galβ4GlcNAβ6)Galβ-;Galβ4GlcNAcβ3-; Galβ4Glcβ-; Galβ3GlcNAcβ-; Galβ3(Fucα4)GlcNAβ-;Fucα2Galβ3(Fucα4)GlcNAcβ-; GalNAcα3(Fucα2)Galβ3(Fucα4)GlcNAcβ-;Galα3(Fucα2)Galβ3(Fucα4)GlcNAcβ-; Galβ4(Fucα3)GlcNAcβ-;Fucα2Galβ4(Fucα3)GlcNAcβ-; NeuAcα2-3Galβ3(Fucα4)GlcNAcβ-;NeuAcα2-3Galβ4(Fucα3)GlcNAcβ-; GalNAβ4(NeuAcα2-3)Galβ4-; Galβ3GalNAcα-;NeuAcα2-3Galβ4-; NeuAcα2-6Galβ4-; Galα4Galβ4-; GalNAcβ3Galα4Galβ4-;Galα4Galβ4GlcNAβ3-; Galβ3GalNAcβ3Galα4-; NeuAcα2-3Galβ3GalNAcβ3Galα4-;Galα3Galβ-; GalNAcα3GalNAcβ3Galα4-; GalNAcβ3GalNAcβ3Galα4-;Galβ1-4GlcNAc; Galβ1-3GlcNAc; SAα2-6Galβ1-4Glc; SAα2-3Galβ1-4Glc;SAα2-6Galβ1-4GlcNAc; SAα2-3Galβ1-4GlcNAc; SAα2-3Galβ1-3GlcNAc;Galβ1-4(Fucα1-3)GlcNAc; Galβ1-3(Fucα1-3)GlcNAc;SAα2-3Galβ1-3(Fucα1-4)GlcNAc; SAα2-3Galβ1-4(Fucα1-3)GlcNAc;Galβ1-4GlcNAcβ1-4GlcNAc; Galβ1-3GlcNAcβ1-4GlcNAc;SAα2-6Galβ1-4GlcNAβ1-4GlcNAc; SAα2-3Galβ1-4GlcNAβ1-4GlcNAc;SAα2-3Galβ1-3GlcNAβ1-4GlcNAc; Galβ1-4(Fucα1-3)GlcNAβ1-4GlcNAc;Galβ1-3(Fucα1-4)GlcNAβ1-4GlcNAc; SAα2-3Galβ1-3(Fucα1-4)GlcNAβ1-4GlcNAc;SAα2-3Galβ1-4(Fucα1-3)GlcNAcβ1-4GlcNAc;SAα2-3Galβ1-3(Fucα1-4)GlcNAβ1-4Gal; SAα2-3Galβ1-4(Fucα1-3)GlcNAβ1-4Gal;SAα2-3Galβ1-4GlcNAβ1-3Galβ1-4(Fucα1-3)GlcNAc;SAα2-6Galβ1-4GlcNAβ1-3Galβ1-4(Fucα1-3)GlcNAc;SAα2-3Galβ1-4(Fucα1-3)GlcNAβ1-3Galβ1-4Glc;SAα2-6Galβ1-4(Fucα1-3)GlcNAβ1-3Galβ1-4Glc;SAα2-3Galβ1-4GlcNAβ1-3Galβ1-4(Fucα1-3)GlcNAβ1-3Galβ1-4Glc;SAα2-6Galβ1-4GlcNAβ1-3Galβ1-4(Fucα1-3)GlcNAβ1-3Galβ1-4Glc;SAα2-3Galβ1-4(Fucα1-3)GlcNAβ1-3Galβ1-4(Fucα1-3)GlcNAβ1-3Galβ1-4Glc;SAα2-6Galβ1-4(Fucα1-3)GlcNAβ1-3Galβ1-4(Fucα1-3)GlcNAβ1-3Galβ1-4Glc;SAα2-3Galβ1-4(Fucα1-3)GlcNAβ1-3Galβ1-4(Fucα1-3)Glc;SAα2-6Galβ1-4(Fucα1-3)GlcNAβ1-3Galβ1-4(Fucα1-3)Glc;SAα2-3Galβ1-4GlcNAβ1-3Galβ1-4(Fucα1-3)GlcNAβ1-3Galβ1-4(Fucα1-3)Glc;SAα2-6Galβ1-4GlcNAβ1-3Galβ1-4(Fucα1-3)GlcNAβ1-3Galβ1-4(Fucα1-3)Glc;SAα2-3Galβ1-4(Fucα1-3)GlcNAβ1-3Galβ1-4(Fucα1-3)GlcNAβ1-3Galβ1-4(Fucα1-3)Glc;SAα2-6Galβ1-4(Fucα1-3)GlcNAβ1-3Galβ1-4(Fucα1-3)GlcNAβ1-3Galβ1-4(Fucα1-3)Glc;SAα2-3Galβ1-3(Fucα1-4)GlcNAc; SAα2-6Galβ1-3(Fucα1-4(GlcNAc;SAα2-3Galβ1-3GlcNAβ1-4Galβ1-4(Fucα1-3)GlcNAc;SAα2-6Galβ1-3GlcNAβ1-4Galβ1-4(Fucα1-3)GlcNAc;SAα2-3Galβ1-3(Fucα1-4)GlcNAβ1-3Galβ1-4(Fucα1-3)GlcNAc;SAα2-6Galβ1-3(Fucα1-4)GlcNAβ1-3Galβ1-4(Fucα1-3)GlcNAc;SAα2-3Galβ1-3(Fucα1-4)GlcNAβ1-3Galβ1-4Glc;SAα2-6Galβ1-3(Fucα1-4)GlcNAβ1-3Galβ1-4Glc;SAα2-3Galβ1-3GlcNAβ1-4Galβ1-4(Fucα1-3)GlcNAβ1-3Galβ1-4Glc;SAα2-6Galβ1-3GlcNAβ1-4Galβ1-4(Fucα1-3)GlcNAβ11-3Galβ1-4Glc;SAβ2-3Galβ1-3(Fucα1-4)GlcNAβ1-3Galβ1-4(Fucα1-3)GlcNAβ1-3Galβ1-4Glc;SAα2-6Galβ1-3(Fucα1-4)GlcNAβ1-3Galβ1-4(Fucα1-3)GlcNAβ1-3Galβ1-4Glc;SAα2-3Galβ1-3(Fucα1-4)GlcNAβ1-3Galβ1-4(Fucα1-3)Glc;SAα2-6Galβ1-3(Fucα1-4)GlcNAβ1-3Galβ1-4(Fucα1-3)Glc;SAα2-3Galβ1-3GlcNAβ1-4Galβ1-4(Fucα1-3)GlcNAβ1-3Galβ1-4(Fucα1-3)Glc;SAα2-6Galβ1-3GlcNAβ1-4Galβ1-4(Fucα1-3)GlcNAβ1-3Galβ1-4(Fucα1-3)Glc;SAα2-3Galβ1-3(Fucα1-4)GlcNAβ1-3Galβ1-4(Fucα1-3)GlcNAβ1-3Galβ1-4(Fucα1-3)Glc;SAα2-6Galβ1-3(Fucα1-4)GlcNAβ1-3Galβ1-4(Fucα1-3)GlcNAβ1-3Galβ1-4(Fucα1-3)Glc;SAα2-3Galβ1-4GlcNAβ1-3Galβ1-3(Fucα1-4)GlcNAc;SAα2-6Galβ1-4GlcNAβ1-3Galβ1-3(Fucα1-4)GlcNAc;SAα2-3Galβ1-4(Fucα1-3)GlcNAβ1-3Galβ1-3(Fucα1-4)GlcNAc;SAα2-6Galβ1-4(Fucα1-3)GlcNAβ1-3Galβ1-3(Fucα1-4)GlcNAc;SAα2-3Galβ1-4GlcNAβ1-3Galβ1-3(Fucα1-4)GlcNAβ1-3Galβ1-4Glc;SAα2-6Galβ1-4GlcNAβ1-3Galβ1-3(Fucα1-4)GlcNAβ1-3Galβ1-4Glc;SAα2-3Galβ1-4(Fucα1-3)GlcNAβ1-3Galβ1-3(Fucα1-4)GlcNAβ1-3Galβ1-4Glc;SAα2-6Galβ1-4(Fucα1-3)GlcNAβ1-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc;SAα2-3Galβ1-4(Fucα1-3)GlcNAβ1-3Galβ1-3(Fucα1-4)Glc;SAα2-6Galβ1-4(Fucα1-3)GlcNAβ1-3Galβ1-3(Fucα1-4)Glc;SAα2-3Galβ1-4GlcNAβ1-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc;SAα2-6Galβ1-4GlcNAcβ1-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc;SAα2-3Galβ1-4(Fucα1-3)GlcNAβ1-3Galβ1-3(Fucα1-4)GlcNAβ1-3Galβ1-4(Fucα1-3)Glc;andSAα2-6Galβ1-4(Fucα1-3)GlcNAβ1-3Galβ1-3(Fucα1-4)GlcNAβ1-3Galβ1-4(Fucα1-3)Glc,where SA is sialic acid.

In a second preferment of the first aspect of the invention F is apeptide. More preferably, F is a peptide that is an oligopeptide. Mostpreferably, F is a peptide selected from the group listed in the Tableof Peptides.

In a third preferment of the first aspect of the invention F is aconjugator. More preferably, F is a conjugator that is biotin. When F isa conjugator that is biotin, the biotin may or may not be conjugated toan avidinylated functional moiety.

Preferably, the method includes the step of coating the surface of thesubstrate with a polymer after the propelling of the droplets of thedispersion of the synthetic construct of the structure F-S-L onto thesurface of the substrate. More preferably, the method includes the stepof coating the surface of the substrate with isobutyl methacrylatepolymer after the propelling of the droplets of the dispersion of thesynthetic construct of the structure F-S-L.

Preferably, when F is a glycan, S is selected from the group consistingof:

where:

-   -   a and b are independently the integer 3, 4 or 5; and    -   R₁ and R₂ are, respectively, O of the glycan and N of the        primary amino of a diacyl or dialkyl-glycerophospholipid or N of        the primary amino of a diacyl or dialkyl-glycerophospholipid and        O of the glycan, or

where:

-   -   M is CH₃ or H;    -   c is the integer 3, 4 or 5;    -   d and e are independently the integer 1 or 2;    -   f is the integer 2, 3 or 4;    -   R₃ is N of the primary amino of a diacyl or        dialkyl-glycerophospholipid; and    -   R₄ is O of the glycan.

Preferably, when F is a peptide, S is selected from the group consistingof:

where:

-   -   M is CH₃ or H;    -   c is the integer 3, 4 or 5;    -   d and e are independently the integer 1 or 2;    -   R₅ is N of the primary amino of a diacyl or        dialkyl-glycerophospholipid; and    -   R₆ is S of the sulfhydryl of an amino acid residue of the        peptide,

where:

-   -   g is a value in the range 6 to 14;    -   R₇ and R₈ are, respectively, N of the amino terminus of the        peptide and N of the primary amino of a diacyl or        dialkyl-glycerophospholipid or N of the primary amino of a        diacyl or dialkyl-glycerophospholipid and N of the amino        terminus of the peptide,        or

where:

-   -   g is a value in the range 6 to 14;    -   h is the integer 1 or 2;    -   R₉ is N of the primary amino of a diacyl or        dialkyl-glycerophospholipid; and    -   R₁₀ is S of the sulfhydryl of an amino acid residue of the        peptide, or        or

-   -   g is a value in the range 6 to 14;    -   R₁₁ and R₁₂ are, respectively, N of the amino terminus of the        peptide and N of the primary amino of a diacyl or        dialkyl-glycerophospholipid or N of the primary amino of a        diacyl or dialkyl-glycerophospholipid and N of the amino,        terminus of the peptide.        [followed by page 11]

Preferably, when F is a conjugator that is biotin, F-S is selected fromthe group consisting of:

where:

-   -   M is CH₃ or H;    -   c is the integer 3, 4 or 5;    -   d and e are independently the integer 1 or 2; and    -   R₁₃ is N of the primary amino of a diacyl or        dialkyl-glycerophospholipid.

F may or may not include an avidinylated functional moiety.

In a second aspect the invention provides a diagnostic test card orstick, microarray or multiwell plate fabricated using the method of thefirst aspect of the invention.

In the description and claims of this specification the followingacronyms, terms and phrases have the meaning provided:

“Belt” means, with reference to inkjet printing, the means of attachmentbetween the printhead and stepper motor.

“Control Circuitry” means, with reference to inkjet printing, that partof the printer that controls the mechanical aspects of operation of theprinter.

“Dispersible in water” means a stable, single phase system is formedwhen the synthetic construct is contacted with water.

“Glycan” means a polysaccharide or oligosaccharide and includes thecarbohydrate portion of a glycoconjugate, such as a glycoprotein,glycolipid, or a proteoglycan.

“Immobilised” means covalently bound to a surface and “immobilising” and“immobilisation” have a corresponding meaning.

“Impact” means, with reference to printing on the surface of asubstrate, a method of printing where in image is created by a printermechanism contacting the surface, e.g. character and dot matrixprinters.

“Ink Cartridge” means with reference to inkjet printing, that part ofthe print head assembly comprising a reservoir for containing ink.

“Inkjet Printer” means a non-impact printer that propels droplets of inkonto the surface of a substrate to create an image consisting of aplurality of dots (typically between 45 and 65 μm in diameter.

“Localised” means associated with a surface by non-covalent interactionsand “localising” and “localisation” have a corresponding meaning.

“Microarray” means a two-dimensional array of small quantities ofbiological material.

“Monolithic” means, with reference to a printhead, the plurality oforifices (nozzles) from which droplets of ink are propelled are formedin a single body of material, e.g. a silicon substrate, by means such asphotolithography or chemical etching.

“Non-Impact” means, with reference to printing on the surface of asubstrate, a method of printing where an image is created without aprinter mechanism contacting the surface, e.g. inkjet and laserprinters.

“Picolitre” means a volume of 10⁻¹² litre (pL).

“Piezoelectric” means, with reference to inkjet printing, the method ofpropelling droplets of ink from the orifices (nozzles) of the printheadby vibration of piezo crystals.

“Polar functional groups” means any one or more of a carbonyl (—C═O),carboxyl (—COOH) or secondary amino (>NH) group.

“Printhead” means, with reference to inkjet printing, that part of theprint head assembly comprising a plurality of orifices (nozzles) fromwhich droplets of ink are propelled.

“Printhead Stepper Motor” means, with reference to inkjet printing, thatpart of the printer that drives the movement of the printhead across thesurface of a substrate.

“Rollers” means, with reference to inkjet printing, a set of rollersoperating to advance the surface of a substrate through the transversepath of the printhead.

“Substrate Feed Stepper Motor” means, with reference to inkjet printing,that part of the printer that drives the rollers to advance the surfaceof the substrate through the transverse path of the printhead.

“Thermal Bubble” means, with reference to inkjet printing, the method ofpropelling droplets of ink from the orifices (nozzles) of the printheadby vaporizing a volume of the ink.

“Spacer” means a chemical moiety distinct from the base (e.g.ethanolamine) of a glycerophospholipid comprising at least three polarfunctional groups.

In the description and claims of this specification the amino acids ofpeptides are identified in accordance with Tables 1 to 4 of Annex C,Appendix 2 of the PCT Administrative Instructions (as in force from Jan.1, 2010).

The use of the terms “first”, “second”, “third”, etc. with reference toelements, features or integers of the subject matter defined in theStatement of Invention and Claims, or with reference to alternativeembodiments or preferments of the invention is intended to distinguishbetween alternatives and is not intended to imply an order of preferenceunless specifically stated.

The invention will now be described with reference to the followingTable of Peptides, embodiments or examples, and the figures of theaccompanying drawings pages.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1. Schematic representation of a conventional inkjet printeradapted for use in a method of fabricating microarrays in accordancewith the method of the invention.

FIG. 2. Diagrammatic illustration of patterning of water dispersiblesynthetic constructs to provide test strips capable of identifying thepresence of a plurality of binding molecules in a test sample.

FIG. 3. Diagrammatic illustration of patterning of water dispersiblesynthetic constructs to provide (A) test strips, and (B) multi-wellmicroplates, capable of use in determining the titre a binding moleculein a test sample.

FIG. 4. Appearance of samples of substrate to which solutions of theaminopropyl derivatives of blood group A (A_(tri)) and blood group B(B_(tri) trisaccharides and the construct A_(tri)-Sp-Ad-DOPE (I) (FSL-A)had been applied following visualisation by: i) anisaldehyde(aluminium-backed silica gel plate); ii) immunostaining(aluminium-backed silica gel plate with plasticizer); iii)immunostaining (aluminium backed C₁₈ derivatised silica gel plate withplasticizer); iv) immunostaining (aluminium backed C₁₈) derivatisedsilica gel without plasticizer); and V) immunostaining (nitrocellulose).

FIG. 5. A multiwell plate fabricated according to the method describedshowing visualisation of anti-A immunoglobulin binding toA_(tri)-sp-Ad-DOPE (I) (FSL-A) applied at increasing densities (quantityper unit area) employing the standard ink cartridge and grey scalesetting (30% “2” to 100% “9’) of an EPSON Stylus™ Colour 460 printer.

SEQ ID Table of Peptides NO:Cys(Xaa)_(z)TrpThrProProArgAlaGlnIleThrGlyTyrLeuThrValGlyLeuThrArgArg  1Cys(Xaa)_(z)TrpThrProProArgAlaGlnIleThrGlyTyrArgLeuThrValGlyLeuThrArgArg 2 Cys(Xaa)_(z)ValMetTyrAlaSerSerGly  3                                                         ValMetTyrAlaSerSerGly(Xaa)_(z)Cys  4                                             AspTyrHisArgValMetTyrAlaSerSerGly(Xaa)_(z)Cys  5                                          ThrAsnGlyGluThrGlyGlnLeuValHisArgPhe(Xaa)_(z)Cys  6                                          ThrAsnGlyGluMetGlyGlnLeuValHisArgPhe(Xaa)_(z)Cys  7                                       AspThrTyrProAlaHisThrAlaAsnGluValSerGlu(Xaa)_(z)Cys  8                                                ThrTyrProAlaHisThrAlaAsnGluVal(Xaa)_(z)Cys  9                                                      ProAlaHisThrAlaAsnGluVal(Xaa)_(z)Cys 10                                                      TyrProAlaHisThrAlaAsnGlu(Xaa)_(z)Cys 11                                                      ThrTyrProAlaHisThrAlaAsn(Xaa)_(z)Cys 12                                                   ThrTyrProAlaHisThrAlaAsnGlu(Xaa)_(z)Cys 13                                                   TyrProAlaHisThrAlaAsnGluVal(Xaa)_(z)Cys 14                                                   ProAlaHisThrAlaAsnGluValSer(Xaa)_(z)Cys 15                                                AspThrTyrProAlaHisThrAlaAsnGlu(Xaa)_(z)Cys 16                                                TyrProAlaHisThrAlaAsnGluValSer(Xaa)_(z)Cys 17                                                SerGlnThrAsnAspLysHisLysArgAsp(Xaa)_(z)Cys 18                                             GlnThrAsnAspLysHisLysArgAspThrTyr(Xaa)_(z)Cys 19      GlnThrAsnAspLysHisLysArgAspThrTyrSerSerGlnThrAsnAspMetHisLysArgAspThrTyr(Xaa)_(z)Cys 20                                             GlnThrAsnAspMetHisLysArgAspThrTyr(Xaa)_(z)Cys 21                                                SerSerGlnThrAsnAspLysHisLysArg(Xaa)_(z)Cys 22                                       SerSerGlnThrAsnAspLysHisLysArgAspThrTyr(Xaa)_(z)Cys 23                                       SerSerGlnThrAsnAspMetHisLysArgAspThrTyr(Xaa)_(z)Cys 24SerSerGlnThrAsnAspLysHisLysArgAspThrTyrSerSerGlnThrAsnAspMetHisLysArgAspThrTyr(Xaa)_(z)Cys 25                                                GlnThrAsnAspLysHisLysArgAspThr(Xaa)_(z)Cys 26                                             SerGlnThrAsnAspLysHisLysArgAspThr(Xaa)_(z)Cys 27                                             ThrAsnAspLysHisLysArgAspThrTyrPro(Xaa)_(z)Cys 28                                                GluGluThrGlyGluThrGlyGlnLeuVal(Xaa)_(z)Cys 29                                                GluGluGluThrGlyGluThrGlyGlnLeu(Xaa)_(z)Cys 30                                                GluThrGlyGluThrGlyGlnLeuValHis(Xaa)_(z)Cys 31                                                   SerProProArgArgAlaArgValThr(Xaa)_(z)Cys 32                              TyrArgTyrArgTyrThrProLysGluLysThrGlyProMetLysGlu(Xaa)_(z)Cys 33                                                      TrpGlnProProArgAlaArgIle(Xaa)_(z)Cys 34                                                ThrIleThrGlyLeuGluProGlyThrGlu(Xaa)_(z)Cys 35

FIG. 6. Template design for use in the fabrication of a multiwell platefor use in quantifying antibody titres.

FIG. 7A. A fabricated multiwell plate employing the template of FIG. 6.

FIG. 7B. An enlargement of one of the wells of the fabricated multiwellplate of FIG. 7A.

FIG. 8A. A fabricated multiwell plate employing a template to identifythe location of each well.

FIG. 8B. An enlargement of one of the wells of the fabricated multiwellplate of FIG. 8A.

FIG. 9. The fabricated multiwell plates used in the detection of bindingmolecules in biological samples.

FIG. 10. Immunostaining with monoclonal antibody of the surface ofsubstrates (silica gel and paper) printed with a dispersion of FSL-A.The identity of the substrate employed is identified by the wordsappearing following immunostaining: silica gel (A), Sapphire Cast Coated(Spicers)(B), Impress Silk (Spicers) (C), Impress Gloss (Spicers)(D),Hello Silk (Spicers)(E), Black Velvet Artboard (F), G-Print Matt (G),Alpine Artboard (H), Superfine Hi Gloss (Spicers)(I and J) and uncoatedpaper (K).

FIG. 11. Immunostaining with monoclonal and polyclonal (serum) antibodyof the surface of substrates (paper) printed with a dispersion of FSL-A.The identity of the substrate employed is identified by the wordsappearing following immunostaining: Sapphire Cast Coated (Spicers)(B),Impress Silk (Spicers) (C), Impress Gloss (Spicers)(D), G-Print Matt(G), Superfine Hi Gloss (Spicers)(J) and uncoated paper (K).

FIGS. 12A and 12B. Immunostaining with alkaline phosphatase conjugatedstreptavidin of the surface of substrates (nitrocellulose and paper)printed with a dispersion of FSL-Biotin. The identity of the substrateemployed is identified by the words appearing following immunostaining:Sapphire Cast Impress Gloss (Spicers)(D), G-Print Matt (G), Superfine HiGloss (Spicers)(J), uncoated paper (K) and nitrocellulose (L).

FIG. 13. The structure of the construct A_(tri)-Sp-Ad-DOPE (I) (FSL-A)printed on paper using a dispersion of the construct according to themethod of the invention.

DETAILED DESCRIPTION

The advantages provided by the invention arise from the favourableworking interrelationship between a combination of features. Firstly,the synthetic constructs of the structure F-S-L are readily dispersiblein water (“water soluble” as defined herein). Secondly, the syntheticconstructs remain localised to the surface of a substrate despitewashing with aqueous solutions. Thirdly, inkjet printer technology hasproven to be readily adaptable as a means of applying the dispersions ofthe synthetic constructs to the surface of the substrate.

Adopting the analogy with conventional inkjet printing the dispersionsof synthetic constructs are used as an “ink” to print on the surface ofa substrate used as “paper”. Indeed it has been discovered that thesynthetic constructs are localised to the surface of paper withsufficient strength that the functional moiety is not washed away duringblocking and washing steps routinely used in diagnostic assays. The useof existing inkjet printer technology permits the numbers of functionalmoieties to be localised to the surface of the substrate with greatercontrol and accuracy. The ability to accurately control both thequantity and location of functional moieties localised to the surface ofa substrate also permits the printing of “images” that improve theaccuracy and reliability of assay results.

The preparation of water dispersible synthetic constructs F-S-L with arange of functional moieties (F) including biotin, glycans and peptidesis described in the specifications accompanying internationalapplication nos. PCT/NZ2005/000052 (publ. no. WO2005/090368),PCT/NZ2006/000245 (publ. no. WO2007/035116) and PCT/NZ2008/000266 (publ.no. WO 2009/048343).

The selection of a spacer (S) provides a synthetic construct that isreadily dispersible in water. It is also apparent that “printed”synthetic constructs are oriented to permit interaction between thefunctional moiety (F) with a putative binding molecule.

The method of the invention provides the advantage that the requirementfor subsequent blocking of unreacted groups on a chemically activatedsurface (cf. chemical immobilisation) is negated. An additionaladvantage is the prospect of eluting binding molecule bound to itstarget functional moiety from the surface by the use of solvents. Theopportunity to then characterise the functional moiety and bindingmolecule arises.

The use of the chemistry employed in the manufacture of conventionalreverse phase media such as C₈, C₁₈, etc. was initially considered to bemost appropriate for the preparation of lipophilic surfaces to which theconstructs could be localised. In this context it should be noted thatthe term “lipophilic” is being used to encompass any chemistry thatprovides a surface with a strong affinity for the lipid (L) of thesynthetic construct. It is to be recognised that some substrates providea lipophilic surface without the requirement for chemical modification,e.g. nitrocellulose (Fukui et al (2005)) and polystyrene (Huang et al(2006)). The term “lipophilic” as used herein is to be understood as afunctional feature. Of particular note in the context of the presentinvention is that both coated and uncoated printer paper have beendemonstrated to provide a suitable “lipophilic” surface.

The monomeric dissociation constant (K_(D)) in a carbohydrate-proteininteraction is typically in the millimolar (mM) range. Carbohydratemediated biological responses often occur through multivalentinteractions on the cell surface in order to achieve high affinity andspecificity (Chung-Yi et al (2009)). It is anticipated that localisingthe functional moieties to the surface of a substrate by the interactionof the lipophilic surface and the lipid moiety of the syntheticconstruct F-S-L permits the functional moieties of a population ofdeposited synthetic constructs to have a greater opportunity toparticipate in multivalent interactions with binding molecules, e.g.glycan binding proteins (GBPS).

The adaptation of existing inkjet printing technology to depositquantities of a dispersion of synthetic construct provides a convenientand cost effective means of fabricating diagnostic test cards andsticks, microarrays and multiwell plates of standard dimensions. Indeedit will be recognised by analogy with conventional colour inkjetprinting that the patterning of deposition is also readily achievable.Chambers containing dispersions of populations of synthetic constructare substituted for the colour cartridges of the inkjet printer. Chambersize and design can be readily optimised for the fabrication ofmicroarrays and use of aqueous dispersions. The inclusion of arelatively volatile solvent in the aqueous dispersion is anticipated tofacilitate fabrication of the microarrays by promoting evaporation ofthe vehicle. However, as conventional inject technology permits thedelivery of droplets of small size the surface area to volume ratioresults in a sufficient rate of evaporation to permit the use of wateras a vehicle for the dispersions.

Printheads of designs adaptable for use in the method of the presentinvention are well described. A description of the adaptation of aninkjet printer for use in the fabrication of microarrays in accordancewith the method of the invention will now be described with reference toFIG. 1 of the accompanying drawings.

FIG. 1 is a side cross-sectional view schematically showing theprinthead (1) of an inkjet printer and the surface of a substrate (2) injuxtaposition. The printhead (1) is mounted on a carriage (3) thatpermits reciprocating motion (4) of the printhead (1) relative to thesurface of the substrate (2).

The printhead (1) comprises a plurality of modules (4 a, 4 b, 4 c)comprising chambers (5 a, 5 b, 5 c), each containing a dispersion of apopulation of synthetic construct F-S-L. Each chamber (5 a, 5 b, 5 c)includes an orifice (nozzle) (6 a, 6 b, 6 c) through which a droplet ofthe dispersion is discharged when a voltage is applied to apiezoelectric assembly (7 a, 7 b, 7 c) in fluid communication with thedispersion.

Each chamber (5 a, 5 b, 5 c) additionally includes a sealable port (8 a,8 b, 8 c) through which the contents of the chamber may be replenishedand a convoluted channel (9 a, 9 b, 9 c) to provide for pressureequalisation subsequent to the discharge of a droplet.

The application of a voltage to each of the piezoelectric assemblies (7a, 7 b, 7 c) is under the control of a controller (10). In turn thecontroller and reciprocating motion of the printhead relative to thesurface of the substrate are under computer control to permit patterningof the surface of the substrate (2).

The application by the controller (10) of a voltage to the piezoelectricassembly (7 a) causes a droplet of predetermined size to be dischargedvia the orifice (6 a) with sufficient momentum to traverse the distanceto the juxtaposed surface (2).

On contact with the lipophilic surface it is anticipated the syntheticconstructs F-S-L will orient so that the lipid moiety (L) is associatedwith the surface. This dynamic process is promoted by evaporation of theaqueous vehicle and will be dependent on ambient conditions oftemperature and pressure as well as droplet volume as well as thepercentage, if any, of co-solvent, e.g. methanol, present in the aqueousvehicle.

It will be apparent from the foregoing description that dropletsconsisting of different populations of synthetic construct may bedeposited in the same discrete area on the surface of the substrate.Microarrays with patterning of this type may be of assistance inidentifying binding molecules that form multivalent interactions with aplurality of receptors present in the glycocalyx of cells.

Similarly it will be apparent from the foregoing description thatdroplets consisting of different populations of synthetic construct maybe deposited in the discrete areas spaced apart on the surface of thesubstrate and at different concentrations. Microarrays with patterningof this type may be of assistance in identifying the avidity andspecificity of binding molecules.

As discussed above the non-covalent localisation of functional moietiesto a surface presents the prospect of eluting the binding molecule boundto its target functional moiety from the surface. The opportunity tocharacterise the functional moiety and binding molecule then arises.

In anticipated embodiments the method is used to conveniently and costeffectively produce diagnostic test cards and strips and arrays of thetype illustrated diagrammatically in FIG. 2 and FIG. 3.

The diagnostic test cards and strips and arrays may be produced in largenumbers with a high degree of reproducibility making them eminentlysuitable for use in inter-laboratory standardisation.

Reliability and ease of use also suggests the use of the diagnostic testcards and strips and arrays in over-the-counter home test kits.

The patterning of the different populations of synthetic construct maybe readily adjusted to correspond to the format of the automated readingdevice where one is to be used.

In use a test sample is contacted with the diagnostic test strip orarray for a predetermined time to allow binding of binding moleculespresent in the test sample to bind to the functional moiety, e.g.glycotope, of the deposited synthetic construct.

The surface of the diagnostic test strip or array is then washed with anaqueous buffer, such washing facilitated by the lipophilicity of thesurface of the test strip or array.

The presence of bound binding molecule may then be detected by use ofdetection systems such as anti-IgG enzyme conjugates that give rise to achromogenic response in the presence of the appropriate reagents.

It will be recognised that the ability to deposit populations ofsynthetic constructs in discreet areas conveniently provides a negativecontrol for the assay to be performed.

By way of illustration, if the assay to be performed is a detection of aparticular binding molecule in a sample fluid by chromogenic means thenon-deposited area in contact with the sample fluid provides thenegative control.

Confirmation of the presence of the binding molecule in the sample fluidis provided by the contrast in chromogenic response between thedeposited and non-deposited (negative control) area.

Similarly, it will be recognised that a positive control may beincorporated by depositing in a discreet area a population of syntheticconstruct comprising a functional moiety (F) known to be present atdetectable levels in all sample fluids to be tested.

Assurance that the assay has been performed correctly is provided by thecontrast in chromogenic response between the deposited (positivecontrol) and non-deposited area.

The discreet area in which the synthetic construct providing for thispositive control is deposited may be delineated so as to provide aconfirmatory indica to the user, such as a tick symbol or smiley face,or readable phrase.

EXAMPLES

Dispersions of the aminopropyl derivatives of blood group Atrisaccharide (A_(tri)-S₁) and blood group B trisaccharide (B_(tri)-S₁)were prepared at a concentration of 0.6 mM in phosphate buffered saline(pH 7.2) (PBS). A solution of the construct A_(tri)-sp-Ad-DOPE (FSL-A)was also prepared at a concentration of 0.6 mM in PBS.

The solutions were applied using a fine tipped artist's paintbrush ontothe surface of each of three substrates:

-   1. Aluminium-backed silica gel thin layer chromatography plates    (Alugram Nano-SIL G silica TLC plate, 0.2 nm Nano siica gel 60,    Macherey-Nagel);-   2. Aluminium-backed C₁₈ derivatised silica gel plates; and-   3. Nitrocellulose membranes.

One of the samples of aluminium-backed silica gel thin layerchromatography plates to which the solution had been applied was sprayedwith a solution of anisaldehyde. The sprayed plate was heated to 200° C.to visualise staining.

The remaining samples of aluminium-backed silica gel thin layerchromatography plates and aluminium-backed C₁₈ derivatised silica gelthin layer chromatography plates to which the solution had been appliedwere immersed in a solution of PLEXIGUM™ P28 (0.5% isobutyl methacrylatepolymer in n-hexane and diethyl ether) for 1 minute and then air dried.

The surface of all samples to which the solutions had been applied werethen immersed in a solution of 2% (w/v) bovine serum albumin (BSA) inPBS prior to being flooded with a dilution of anti-A immunoglobulin(EPICLONE™ monoclonal, CSL Limited).

The flooded surfaces of the substrates were then washed with PBS priorto being flooded with a 1:400 dilution of alkaline phosphataseconjugated sheep anti-mouse immunoglobulin (Chemicon) for 30 minutes.The flooded surfaces of the substrates were then washed with PBSfollowed by a washing of substrate buffer (100 mM Tris, 100 mM NaCl, 50mM MgCl₂, pH 9.5).

The substrate buffer washed samples were then flooded with a 1:55dilution of chromogenic substrate (18.75 mg/mL nitro blue tetrazoliumchloride and 9.4 mg/mL 5-bromo-4-chloro-3-indolyl phosphate, toluidinesalt)(NBTC-BCIP) for 15 minutes. The appearance of the samples followingincubation with substrate is provided in FIG. 4.

Fabrication of Multiwell Plates

Thirty two holes of 7 mm diameter were cut in a 85×63×3 mm planar pieceof acrylic in a 4×8 matrix so as to correspond with the positions ofhalf of the wells of a standard multiwell microplate. The upper surfaceof the planar piece of acrylic was also engraved with letters along thelong edge and numbers along the short edge so as to allow each hole inthe matrix to be uniquely identified by a two character alphanumericcode. Employing the same template used to direct laser cutting of theplanar piece of acrylic, a solution of the construct A_(tri)-S₁-Ad-DOPE(FSL-A) at a concentration of 1 mg/mL in water was printed onto thesurface of an aluminium-backed silica gel plate.

The solution was loaded into the ink cartridge of an EPSON STYLUS™Colour 460 piezoelectric inkjet printer. A concentration of 0.1% (w/v)bromophenol blue was included to assist in visualising the printed area.The numerals “2” to “9” were printed using the same solution applied atincreasing densities (quantity per unit area) corresponding to the greyscale settings of the printer. The alphanumeric character “2” wasprinted at a grey scale setting of 30% through to the alphanumericcharacter “9” printed at a grey scale setting of 100%. The printed platewas washed by placing in a beaker of deionised water for a period of 20minutes and then air dried. The air dried printed plate was thenimmersed in PLEXIGUIT™ P28 for a period of 1 minute before being airdried for a second time. The laser cut planar piece of acrylic was thenadhered to the surface of the aluminium-backed painted silica gel plateusing multipurpose adhesive. A fabricated 4×8 well microplate wasthereby prepared.

Detection of Binding Molecule

A 100 μL volume of a 2% (w/v) solution of BSA in PBS was dispensed intoeach well of the fabricated microplate. The plates were incubated for 30minutes before aspirating the solution of BSA from each well andrinsing. A 100 μL volume of a 1:4 dilution of mouse anti-Aimmunoglobulin was then dispensed into each well and the plate incubatedfor 30 minutes before rinsing each well with PBS. A 100 μL volume of a1:400 dilution of anti-mouse immunoglobulin was then dispensed into eachwell and the plate incubated for 30 minutes before washing each wellwith substrate buffer. A 100 μL volume of a 1:55 dilution of chromogenicsubstrate was then dispensed into each well and the plates incubated for50 minutes. Each well was finally washed with deionised water and theplate air dried. The air dried plate is presented in FIG. 5.

The method of fabricating described provides for the convenientmanufacture of multiwell plates for simultaneous qualitative andquantitative assessment of binding molecules.

Template Design

Different designs of templates for use in the microarray formats canconveniently made in accordance with user requirements employingstandard word processing or drawing software packages as illustrated inFIGS. 2, 3 and 5 to 9 of the drawings pages.

Quantitative Antibody Testing

By way of illustration of a template design FIG. 6 provides the designof a template for use in quantifying antibody titres. The template isdimensioned to correspond to the dimensions of a standard multiwellmicroplate. The fabricated multiwell plate may therefore be handled byexisting dispensers, washers and camera-based readers of multiwellmicroplates.

A well of the template corresponds to two wells of a standard multiwellmicroplate. The two wells on the right of the template may be employedas control wells. The alphanumeric characters and other symbols shown inthe template are engraved in the acrylic and the wells are cut out usinga laser. The piece of planar acrylic produced is then adhered to aprinted silica gel plate.

Solutions of constructs F-S-L are printed employing the same template inwhich the bars of increasing colour-density correspond to increasingdensities (quantity per unit area) of construct applied. It will berecognised that the standard gray scale settings of the printer may beemployed to provide these increasing densities.

In the template the printing of two constructs is presented. Theconstructs F-S-L each comprise a different ligand (F) for one or morebinding proteins. The binding specificity of one or more bindingproteins in the sample may therefore be conveniently assessed.

In the template the two constructs are printed as bars of decreasingdensity and increasing length.

By the use of the template (and others of comparable design)quantitative assessments of binding may be made in a single well of amultiwell microplate. An internal control (background signal) isprovided by the unprinted region outside the discrete area on thesurface of the substrate to which the constructs have been applied.

The design of the template also permits monitoring of assays and aconvenient means to identify the optimum time to terminate incubations.The controls wells (circled in FIG. 6) have only one of the twoconstruct per well, but printed to provide ladders of increasing densitygoing in both directions. When the middle band appears and a whole lineacross the well is visible the optimum time to terminate the incubationis indicated.

A fabricated microwell plate employing the design of templateillustrated in FIG. 6 and used to determine the titre of antibodyaccording to the method described above is illustrated in FIGS. 7A and7B.

Well Identification

By way of further illustration in circumstances where unequivocalidentification of a well in which a positive reaction has occurred isrequired, a template of the design incorporated into the fabricatedmultiwell plate illustrated in FIGS. 8A and 8B may be employed.

The alphanumeric combination of characters that appear in wells in whicha positive reaction has occurred, unequivocally identify the wellwithout reliance on the user correctly determining the coordinates ofthe well location. The likelihood of user error in manual operations isthereby greatly reduced.

Detection of Binding Molecule in a Biological Sample

To confirm the utility of the fabricated microwell plate in thedetection of binding molecules in biological samples the production ofanti-A antibody in the serum of mice was elicited by immunisation with Asubstance saliva. Individual mice were immunised 2, 3 or 4 times with Asubstance saliva over a three week period. Naïve mice having had noimmunisation were used as a control. Elicitation of anti-A antibody inthe sera of immunised mice was confirmed by transfusion of modified redblood cells (“kodecytes”) according to the methods described ininternational application no. PCT/NZ2009/000209. A 32 well microtiterplate was fabricated according to the method described above using adesign of template in which the alphanumeric symbol “A” was printed in alocation corresponding to the base of each well. Each well was filledwith 2% BSA in PBS and incubated at room temperature for at least onehour. Samples of sera collected from immunised mice were diluted 4-foldin 2% BSA in PBS. The 2% BSA in PBS was removed from the wells followingincubation and the diluted samples of sera introduced into individualwells using a pipette. The plates were then incubated at roomtemperature for at least 90 minutes. Following incubation the sampleswere removed from each well and the plate washed several times with PBS.Excess PBS was removed by blotting of the surface of the plate and eachwell then filled with a 400-fold dilution of mouse anti-Ig antibodyconjugated with alkaline phosphatase. The plates were then incubated foran hour before removing the antibody conjugate solution and washingseveral times with PBS. The plate was then washed several times withsubstrate buffer before filling each well of the plate with thechromogenic substrate NBTC-BCIP. The plates were then incubated for 15to 20 minutes until the printed alphanumeric character appeared. Thesubstrate was then removed and the plate washed under a gentle stream ofdeionised water and dried. The wells to which anti-A antibody containingsamples were introduced were clearly identifiable as illustrated in FIG.9. It will be recognised that alternative template designs may bedeveloped for the assessment of binding to multiple constructs and therequired multiwell microplates conveniently fabricated according to themethod described.

Printing of Paper

The ability of standard printing papers to serve as the substrate foruse in the method of the invention was evaluated. Solutions of theconstruct A_(tri)-sp-Ad-DOPE (FSL-A) and a solution of the aminopropylderivative of the A trisaccharide (A_(tri)-sp-NH₂) were printed on tovarious types of commercially available printing papers as previouslydescribed. An ink jet printer (EPSON STYLUS™ T21) with refillablecartridges modified to hold a smaller volume was employed. The constructA_(tri)-sp-Ad-DOPE (FSL-A) and the aminopropyl derivative of the Atrisaccharide (A_(tri)-sp-NH₂) were prepared as solutions at aconcentration of 6 mM. Each one of the solutions was used to fillseparate modified cartridges permitting both solutions to be printed atthe same time on the same sample of paper. To facilitate identificationand as an illustration of one of the advantages of the invention theidentification of the solution and trade name of the paper employed asthe sample were printed. Following printing of the two solutions eachsample of paper was blocked with a 2% (w/v) solution of BSA andimmunostained with monoclonal anti-A and then anti-mouse IgG conjugatedto alkaline phosphatase and the chromogenic substrate NBT-BCIP. Theimmunostained samples of printed paper are presented in FIG. 10. It willbe observed that there was no immunostaining of the sample of printedpaper in the region where the aminopropyl derivative of the Atrisaccharide (A_(tri)-sp-NH₂) was printed. It is assumed that theaminopropyl derivative of the A trisaccharide (A_(tri)-sp-NH₂) waswashed away during the blocking step and immunostaining procedure.Although the majority of the papers employed in the study were coatedpapers it was also demonstrated that normal uncoated paper could alsoserve as a suitable substrate.

Biological Sample

Samples of printed papers were prepared as described under the precedingheading. On this occasion, polyclonal human blood group O serum andmouse anti-human Ig were employed in the immunostaining procedure. Aconcentration of 0.05% w/v bromophenol blue was included in thesolutions of the construct A_(tri)-sp-Ad-DOPE (FSL-A) to permitvisualisation of the printed solutions. The dye was removed followingthe initial step of printing by placing the samples of printed paper ina beaker of deionised water for 15 minutes followed by air drying. Thedried printed samples of paper were then blocked with a solution of BSAfor 30 minutes as previously described. The surface of each sample ofprinted paper was then flooded with a 1 in 4 dilution of either anti-Amonoclonal antibody or the O serum for 60 minutes at room temperature.The flooded surface of the printed sample of paper was then washedrepeatedly by flooding the surface of each sample of printed paper withPBS for 20 seconds and rinsing with PBS. A comparison of the appearanceof the printed samples of paper at each step of the procedure ispresented in FIG. 11. It will be observed that no immunostaining in theregion where the aminopropyl derivative of the A trisaccharide(A_(tri)-sp-NH₂) was printed occurs. The construct A_(tri)-sp-Ad-DOPE(I) (FSL-A) is detected following immunostaining with either monoclonalor polyclonal (serum) antibodies. Certain of the coated papers wereobserved to provide great contrast in the immunostaining procedure whenpolyclonal (serum) antibodies were employed.

Printing of FSL-Biotin

A dispersion of the construct FSL-Biotin at a concentration of 1 mg/ml(6 mM) in PBS and containing 0.05% (w/v) bromophenol blue was prepared.A volume of the solution was injected into the modified refillablecartridge of a piezoelectric printer (EPSON™ Stylus T21). The dispersionof the construct FSL-Biotin was printed onto samples of coated papersand uncoated paper. The dispersion of the construct FSL-Biotin was alsoprinted onto aluminium-backed silica TLC plates (0.2 mm Nano silica gel60, Macherey-Nagel) and nitrocellulose membranes (0.02 μL pore size,Invitrogen).

The samples of printed paper were placed in a beaker of delonised waterfor 15 minutes to remove the bromophenol blue dye and then air dried.The sample of aluminium backed silica gel TLC plate was immersed in asolution of 0.5% (w/v) polyisobutylmethacrylate in n-hexane and diethylether (PLEXIGUM P28) for one minute and then air dried. Neither thesamples of printed paper nor sample of printed nitrocellulose wassubjected to this treatment prior to immunostaining. For immunostainingthe samples were first blocked by flooding the surface with a solutionof 2% (w/v) BSA in PBS. The surface of each sample was then flooded withstreptavidan-alkaline phosphatase conjugate (sigma) at a concentrationof 2 μg/ml for 30 minutes at room temperature. The samples were thenwashed with PBS by flooding the surface of the membranes with PBS for 20seconds and repeating 6 times with fresh PBS for each washing step. Theprinted samples were then washed with substrate buffer (100 mM Tris, 100mM NaCl, 50 mM MgCl₂, pH 9.5) prior to flooding the surface of eachsample with NBT/BCIP substrate (18.75 mg/ml nitro blue tetrazoliumchloride and 9.4 mg/ml 5-bromo-4-chloro-3-indoyl-phosphate, toluidinesalt in 67% (v/v) DMSO (Roche) diluted 50-fold in substrate buffer andincubated for 50 minutes at room temperature. The printed samples wererinsed with deionised water to stop the chromogenic reaction. Theresults of immunostaining each of the samples are presented in FIGS. 12Aand 12B.

Although the invention has been described by way of exemplaryembodiments it should be appreciated that variations and modificationsmay be made without departing from the scope of the invention. Whereknown equivalents exist to specific features, such equivalents areincorporated as if specifically referred to in this specification.

REFERENCES

-   Blixt et al (2004) Printed covalent glycan array for ligand    profiling of diverse glycan binding proteins PNAS, 101(49),    17033-17038.-   Bovin and Huflejt (2008) Unlimited glycochip Trends Glycosci.    Glycotechnol. 20(115), 245-258.-   Campanero-Rhodes et al (2007) N-glycolyl GM1 ganglioside as a    receptor for simian virus 40 Journal of Virology, 81(23),    12846-12858.-   Chai et al (2003) Neoglycolipid technology: Deciphering information    content of glycome Methods in Enzymol., 362, 160-195.-   Chai et al (2004) Products and methods U.S. patent application Ser.    No. 10/855,072 (publ. no. US 2004/0259142 A1).-   Chung-Yi et al (2009) New development of glycan arrays Org. Biomol.    Chem., 7, 2247-2254.-   Feizi et al (2003) Neoglycolipids: identification of functional    carbohydrate epitopes Carbohydrate-Based Drug Discovery, Volume 2,    747-760. Editor(s): Wong, Chi-Huey. Publisher: Wiley-VCH Verlag GmbH    & Co. KGaA, Weinheim, Germany.-   Feizi and Chai (2004) Innovation: Oligosaccharide microarrays to    decipher the glycocode Nature Reviews Molecular Cell Biology, 5(7),    582-588.-   Feizi (2006) Oligosaccharide microarrays to decipher the glyco code    Abstracts of Papers, 231st ACS National Meeting, Atlanta, Ga.,    United States, Mar. 26-30, 2006, CARB-016 Publisher: American    Chemical Society, Washington, D.C.-   Fukui et al (2002) Oligosaccharide microarrays for high-throughput    detection and specificity assignments of carbohydrate protein    interactions Nat. Biotechnol., 20(10), 1011-1017.-   Fukui (2003) Carbohydrate microarray: a sweet spot for deciphering    the information embedded in oligosaccharide structures Seikagaku,    75(12), 1545-1550.-   Huang et al (2006a) Fabrication and application of neoglycolipid    arrays in a microtitre plate Bioorg. Med. Chem. Lett., 16,    2031-2033.-   Huang et al (2006b) Structure-function relations of carbohydrates by    neoglycolipid arrays Applied Biochemistry and Biotechnology, 133(3),    211-215.-   Liu et al (2006) Preparation of neoglycolipids with ring-closed    cores via chemoselective oxime-ligation for microarray analysis of    carbohydrate-protein interactions Methods in Enzymol.,    415(Glycobiology), 326-340.-   Liu et al (2007) Neoglycolipid Probes Prepared via Oxime Ligation    for Micro-array Analysis of Oligosaccharide-Protein Interactions    Chemistry & Biology, 14(7), 847-859.-   Palma et al (2006) Ligands for the β-Glucan Receptor, Dectin-1,    Assigned Using “Designer” Microarrays of Oligosaccharide Probes    (Neoglycolipids) Generated from Glucan Polysaccharides Journal of    Biological Chemistry, 281(9), 5771-5779.-   Shin et al (2005) Carbohydrate microarrays: An advanced technology    for functional studies of glycans Chem. Eur. J., 11, 2894-2901.-   Yamaguchi et al (2006) Detection of oligosaccharide ligands for    Hepatocyte growth factor/Scatter factor (HGF/SF), Keratinocyte    growth factor (KGF/FGF-7), RANTES and Heparin cofactor II by    neoglycolipid microarrays of glycosaminoglycan-derived    oligosaccharide fragments Glycoconjugate Journal, 23(7/8), 513-523.

1-39. (canceled)
 40. A method of localising a functional moiety (F) toat least one discrete area on a surface of a substrate including thestep of propelling droplets of a dispersion of a synthetic construct ofthe structure F-S-L from a plurality of orifices located in a print headof an inkjet printer onto the surface where S is a spacer selected toprovide a construct that is dispersible in water at a temperature of 25°C. in the absence of organic solvents or detergents and L is a diacyl-or dialkyl lipid.
 41. The method of claim 40 where the method includesthe step of coating the surface of the substrate with a polymer afterthe propelling of the droplets of the dispersion onto the surface. 42.The method of claim 41 where the polymer is isobutyl methacrylate. 43.The method of claim 42 where the substrate is paper.
 44. The method ofclaim 40 where the at least one discrete area is in the shape of asymbol readable by optical character recognition (OCR) apparatus. 45.The method of claim 44 where the at least one discrete area is a patterncomprising a combination of indicia in which the dispersion of asynthetic construct is present at different densities.
 46. The method ofclaim 40 where the concentration of the synthetic construct in thedispersion is 1 μmolar (μM) to 10 mmolar (mM).
 47. The method of claim46 where the concentration of the synthetic construct in the dispersionis 10 μM to 10 mM.
 48. The method of claim 47 where the concentration ofthe synthetic construct in the dispersion is 0.1 to 10 mM.
 49. Themethod of claim 40 where L is a glycerophospholipid.
 50. The method ofclaim 49 where L is a phosphatidylethanolamine.
 51. The method of claim50 where L is 1,2-O-dioleoyl-sn-glycero-3-phosphatidylethanolamine. 52.The method of claim 40 where F is biotin.
 53. The method of claim 52where F-S is selected from the group consisting of:

where: M is CH₃ or H; c is the integer 3, 4 or 5; d and e areindependently the integer 1 or 2; and R₁₃ is N of the primary amino of adiacyl or dialkyl-glycerophospholipid.
 54. The method of claim 40 whereF is an oligosaccharide.
 55. The method of claim 54 where F is anoligosaccharide selected from the group consisting of:GalNAcα3(Fucα2)Galβ3-; Galα3(Fucα2)Galβ3-; GalNα3(Fucα2)Galβ3-;Fucα2Galβ3-; Galβ4GlcNAcβ3(Galβ4GlcNAcβ6)Galβ3-; Galβ4GlcNAcβ3-;Galβ4Glcβ-; Galβ3GlcNAcβ-; Galβ3(Fucα4)GlcNAcβ-;Fucα2Galβ3(Fucα4)GlcNAcβ-; GalNAcα3(Fucα2)Galβ3(Fucα4)GlcNAcβ-;Galα3(Fucα2)Galβ3(Fucα4)GlcNAcβ-; Galβ4(Fucα3)GlcNAcβ-;Fucα2Galβ4(Fucα3)GlcNAcβ-; NeuAcα2-3Galβ3(Fucα4)GlcNAcβ-;NeuAcα2-3Galβ4(Fucα3)GlcNAcβ-; GalNAcβ4(NeuAcα2-3)Galβ4-; Galβ3GalNAcα-;NeuAcα2-3Galβ4-; NeuAcα2-6Galβ4-; Galα4Galβ4-; GalNAcβ3Galα4Galβ4-;Galα4Galβ4GlcNAcβ3-; Galβ3GalNAcβ3Galα4-; NeuAcα2-3Galβ3GalNAcβ3Galα4-;Galα3Galβ3-; GalNAcα3GalNAcβ3Galα4-; GalNAcβ3GalNAcβ3Galα4-;Galβ1-4GlcNAc; Galβ1-3GlcNAc; SAα2-6Galβ1-4Glc; SAα2-3Galβ1-4Glc;SAα2-6Galβ1-4GlcNAc; SAα2-3Galβ1-4GlcNAc; SAα2-3Galβ1-3GlcNAc;Galβ1-4(Fucα1-3)GlcNAc; Galβ1-3(Fucα1-3)GlcNAc;SAα2-3Galβ1-3(Fucα1-4)GlcNAc; SAα2-3Galβ1-4(Fucα1-3)GlcNAc;Galβ1-4GlcNAcβ1-4GlcNAc; Galβ1-3GlcNAcβ1-4GlcNAc;SAα2-6Galβ1-4GlcNAcβ1-4GlcNAc; SAα2-3Galβ1-4GlcNAcβ1-4GlcNAc;SAα2-3Galβ1-3GlcNAcβ1-4GlcNAc; Galβ1-4(Fucα1-3)GlcNAcβ1-4GlcNAc;Galβ1-3(Fucα1-4)GlcNAcβ1-4GlcNAc;SAα2-3Galβ1-3(Fucα1-4)GlcNAcβ1-4GlcNAc;SAα2-3Galβ1-4(Fucα1-3)GlcNAcβ1-4GlcNAc;SAα2-3Galβ1-3(Fucα1-4)GlcNAcβ1-4Gal;SAα2-3Galβ1-4(Fucα1-3)GlcNAcβ1-4Gal;SAα2-3Galβ1-4GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAc;SAα2-6Galβ1-4GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAc;SAα2-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc;SAα2-6Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc;SAα2-3Galβ1-4GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc;SAα2-6Galβ1-4GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc;SAα2-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc;SAα2-6Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc;SAα2-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc;SAα2-6Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc;SAα2-3Galβ1-4GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc;SAα2-6Galβ1-4GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc;SAα2-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc;SAα2-6Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc;SAα2-3Galβ1-3(Fucα1-4)GlcNAc; SAα2-6Galβ1-3(Fucα1-4(GlcNAc;SAα2-3Galβ1-3GlcNAcβ1-4Galβ1-4(Fucα1-3)GlcNAc;SAα2-6Galβ1-3GlcNAcβ1-4Galβ1-4(Fucα1-3)GlcNAc;SAα2-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAc;SAα2-6Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAc;SAα2-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc;SAα2-6Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc;SAα2-3Galβ1-3GlcNAcβ1-4Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc;SAα2-6Galβ1-3GlcNAcβ1-4Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc;SAβ2-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc;SAα2-6Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc;SAα2-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc;SAα2-6Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc;SAα2-3Galβ1-3GlcNAcβ1-4Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc;SAα2-6Galβ1-3GlcNAcβ1-4Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc;SAα2-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc;SAα2-6Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc;SAα2-3Galβ1-4GlcNAcβ1-3Galβ1-3(Fucα1-4)GlcNAc;SAα2-6Galβ1-4GlcNAcβ1-3Galβ1-3(Fucα1-4)GlcNAc;SAα2-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-3(Fucα1-4)GlcNAc;SAα2-6Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-3(Fucα1-4)GlcNAc;SAα2-3Galβ1-4GlcNAcβ1-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc;SAα2-6Galβ1-4GlcNAcβ1-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc;SAα2-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc;SAα2-6Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc;SAα2-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-3(Fucα1-4)Glc;SAα2-6Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-3(Fucα1-4)Glc;SAα2-3Galβ1-4GlcNAcβ1-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc;SAα2-6Galβ1-4GlcNAcβ1-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc;SAα2-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc;andSAα2-6Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc,where SA is sialic acid.
 56. The method of claim 54 where S is selectedfrom the group consisting of:

where: a and b are independently the integer 3, 4 or 5; and R₁ and R₂are, respectively, O of the glycan and N of the primary amino of adiacyl or dialkyl-glycerophospholipid or N of the primary amino of adiacyl or dialkyl-glycerophospholipid and O of the glycan, or

where: M is CH₃ or H; c is the integer 3, 4 or 5; d and e areindependently the integer 1 or 2; f is the integer 2, 3 or 4; R₃ is N ofthe primary amino of a diacyl or dialkyl-glycerophospholipid; and R₄ isO of the glycan.
 57. The method of claim 40 where F is an oligopeptide.58. The method of claim 57 where F is an oligopeptide consisting of asequence selected from the group consisting of SEQ ID NOs 1 to
 35. 59.The method of claim 57 where S is selected from the group consisting of:

where: M is CH₃ or H; c is the integer 3, 4 or 5; d and e areindependently the integer 1 or 2; R₅ is N of the primary amino of adiacyl or dialkyl-glycerophospholipid; and R₆ is S of the sulfhydryl ofan amino acid residue of the peptide,

where: g is a value in the range 6 to 14; R₇ and R₈ are, respectively, Nof the amino terminus of the peptide and N of the primary amino of adiacyl or dialkyl-glycerophospholipid or N of the primary amino of adiacyl or dialkyl-glycerophospholipid and N of the amino terminus of thepeptide, or

where: g is a value in the range 6 to 14; h is the integer 1 or 2; R₉ isN of the primary amino of a diacyl or dialkyl-glycerophospholipid; andR₁₀ is S of the sulfhydryl of an amino acid residue of the peptide, oror

g is a value in the range 6 to 14; R₁₁ and R₁₂ are, respectively, N ofthe amino terminus of the peptide and N of the primary amino of a diacylor dialkyl-glycerophospholipid or N of the primary amino of a diacyl ordialkyl-glycerophospholipid and N of the amino terminus of the peptide.60. A diagnostic test card or stick, microarray or multiwell platefabricated using the method of claim 40.